There are differences in electrical potential and chemical composition between the fluid compartments of the inner ear and the inside of its cells. The resulting electrochemical gradients act as a battery to power a membrane-based motor in the outer hair cell. The motor differs from other biological motors because it converts a difference in electrical potential directly into a mechanical force rather than utilizing chemical energy stored within cells. As a result it can generate force at very high frequencies and the outer hair cell uses its electric motor to enhance the sensitivity and frequency selectivity of hearing. The goal of our multidisciplinary team is to understand the biological and physical basis of the outer hair cell motor at the molecular and cellular level. The mechanism resides in the cell's novel lateral wall, a 100 nanometer thick, three-layer structure composed of two membranes with a cytoskeletal network sandwiched between them. Optimal performance of the motor requires the membrane protein prestin, intracellular chloride, low membrane cholesterol and an intact lateral wall. The specific objectives of this project period are to evaluate each of these features. Prestin belongs to a family of membrane proteins that facilitate the movement of anions (negatively charged ions such as chloride) through the cell membrane. The structure and function of prestin are analyzed and the contributions of the lateral wall membranes to the modulation and maintenance of the electrochemical gradients necessary for cell function are examined. Coordinated theoretical and experimental approaches are used to identify how prestin works together with anions, other membrane components (including cholesterol) and membrane reactive agents (such as quinine) to modulate the movement of electrical charge into and out of the membrane. Molecular and cellular biology, bioinformatics, biophysics, neuroscience, chemical physics, and bioengineering approaches will all be used. These studies will reveal the frequency response of the motor and show how sound is amplified at high frequencies in the inner ear. Understanding prestin function will provide new information on the anion transport properties of its close family members, which are implicated in diseases associated with anion homeostasis within the lung, pancreas, kidney, colon and inner ear. Elucidation of the biological and physical principles underlying the outer hair cell motor will contribute to the emerging field of biological nanotechnology. These studies may lead to improved therapeutic interventions for the hearing impaired. The studies are directly applicable to understanding why deafness follows the loss of outer hair cells; our results will explain how inner ear vibrations are enhanced at high frequencies to improve hearing. Understanding how the membrane protein prestin functions will provide new information on the anion transport properties of its close family members including pendrin, a mutation of which also leads to hearing loss. In addition to potential benefits to the hearing impaired, elucidation of the physical principles underlying the outer hair cell motor will contribute to the emerging field of biological nanotechnology.